Escherichia coli metabolism under short-term repetitive substrate dynamics: adaptation and trade-offs

Abstract

Background: Microbial metabolism is highly dependent on the environmental conditions. Especially, the substrate concentration, as well as oxygen availability, determine the metabolic rates. In large-scale bioreactors, microorganisms encounter dynamic conditions in substrate and oxygen availability (mixing limitations), which influence their metabolism and subsequently their physiology. Earlier, single substrate pulse experiments were not able to explain the observed physiological changes generated under large-scale industrial fermentation conditions.

Results: In this study we applied a repetitive feast-famine regime in an aerobic Escherichia coli culture in a time-scale of seconds. The regime was applied for several generations, allowing cells to adapt to the (repetitive) dynamic environment. The observed response was highly reproducible over the cycles, indicating that cells were indeed fully adapted to the regime. We observed an increase of the specific substrate and oxygen consumption (average) rates during the feast-famine regime, compared to a steady-state (chemostat) reference environment. The increased rates at same (average) growth rate led to a reduced biomass yield (30% lower). Interestingly, this drop was not followed by increased by-product formation, pointing to the existence of energy-spilling reactions. During the feast-famine cycle, the cells rapidly increased their uptake rate. Within 10 s after the beginning of the feeding, the substrate uptake rate was higher (4.68 μmol/g_CDW/s) than reported during batch growth (3.3 μmol/g_CDW/s). The high uptake led to an accumulation of several intracellular metabolites, during the feast phase, accounting for up to 34% of the carbon supplied. Although the metabolite concentrations changed rapidly, the cellular energy charge remained unaffected, suggesting well-controlled balance between ATP producing and ATP consuming reactions.

Conclusions: The adaptation of the physiology and metabolism of E. coli under substrate dynamics, representative for large-scale fermenters, revealed the existence of several cellular mechanisms coping with stress. Changes in the substrate uptake system, storage potential and energy-spilling processes resulted to be of great importance. These metabolic strategies consist a meaningful step to further tackle reduced microbial performance, observed under large-scale cultivations.

Keywords: Dynamic metabolic responses; Energy homeostasis; Escherichia coli; Feast–famine; Substrate dynamics.

Fig. 1

Schematic representation of the feast–famine setup used in this work. The medium, containing glucose as a substrate, was fed block-wise (20 s on, 380 s off) through a head-plate port. A constant volume was maintained by weight control. Successive cycles run for about 200 h in total

Fig. 2

Measured concentrations and calculated rates during the feast–famine cycle (s), approximately eight generations after the beginning of the regime. a Residual glucose concentration (mM), quantified by GC–MS/MS. b Dissolved oxygen concentration (%) in the broth, raw data (black) and calculated, eliminating delays of the used Clark probe (blue). c Measurements of oxygen content in offgas (%). d Measurements of carbon dioxide content in offgas (%). e Calculated oxygen uptake rate (${\text{mmol}}_{{\text{O}}_2}$/g_CDW/h) based on the headspace and tubing offgas delays. f Calculated carbon dioxide production rate (${\text{mmol}}_{{\text{CO}}_2}$/g_CDW/h) based on the headspace, tubing offgas delays and bicarbonate in the broth. g Respiratory quotient (RQ) over time derived from the calculated ${\text{q}}_{{\text{O}}_2}$ and ${\text{q}}_{{\text{CO}}_2}$. Data of 16 successive cycles are overlapped for DO, O₂ and CO₂ (b–d). The pink area in the plots represents the substrate feast phase. Green vertical dashed lines show the end of the feeding (20 s)

Fig. 3

Glucose concentration and uptake over time. a Residual glucose concentration in mM (black dots) and the PWA fitted profile (blue line) over one cycle time (s). b Glucose uptake rate (− q_glc) in μmoles_glc/g_CDW/s over one cycle time (s). The red dots represent the breakpoints. Note that for the rate only the first 110 s are shown. For t > 110 s, the flux was zero until the end of the cycle. Green vertical dashed lines show the end of the feeding (20 s) and the horizontal dotted lines represent the average steady-state levels

Fig. 4

Network model used for the flux balance analysis. Metabolite names are shown in yellow text boxes. Under each metabolite, its intracellular concentration (μmol/g_CDW) (extracellular only for glucose) over time (s) is shown. Black dots represent the measurements, the red line is the PWA fitted line and black dashed lines represent the average steady-state levels. Green vertical dashed lines show the end of the feeding (20 s). The pink area represents the substrate feast phase. The blue line plots show the FBA estimated flux profiles in μmol_{reaction_substrate}/g_CDW/s, where the blue dots are the values at the breakpoints. Fluxes are shown up to 110 s and they were all zero afterwards until the end of the cycle

Fig. 5

Intracellular concentrations (μmol/g_CDW) of TCA metabolites (a-ketoglutarate, malate and fumarate), over a feast–famine cycle(s). Black horizontal dashed lines represent the average steady-state levels. Green vertical dashed lines show the end of the feeding (20 s). The pink area represents the substrate feast phase

Fig. 6

Intracellular concentrations (μmol/g_CDW) of nucleotides, as well as the adenylate energy charge (AEC), over a feast–famine cycle(s). Black horizontal dashed lines represent the average steady-state levels. Green vertical dashed lines show the end of the feeding (20 s). The pink area represents the substrate feast phase

Fig. 7

Intracellular metabolites and carbon distribution over time. Top: Total amount of intracellular metabolites measured (in μCmol/g_CDW) over a feast–famine cycle (s). The black horizontal dashed line represents the average steady-state levels. Bottom: The carbon distribution (% of the total intracellular metabolome measured) in metabolites of different categories/pathways, over a feast–famine cycle(s). Green vertical dashed lines show the end of the feeding (20 s). The pink area represents the substrate feast phase. The detailed list of metabolites for each category can be found in Additional file 1: S10